How Protein Expression in Bacteria Works

Modern biotechnology leverages bacteria as microscopic factories to produce specific proteins they would not naturally create. This process, known as protein expression, has wide-ranging impacts, allowing scientists to generate large quantities of a single protein for therapeutic, industrial, or research purposes. By harnessing their cellular machinery, complex proteins from other species, including humans, can be manufactured efficiently.

Core Components of Bacterial Expression Systems

The bacterium Escherichia coli (E. coli) is the workhorse for this process. Scientists favor it because its genetics are well-understood, it reproduces quickly, and its growth materials are inexpensive. To produce a foreign protein like human insulin, its genetic instructions—the gene of interest—must be introduced into the bacterium.

This gene is carried into the bacteria using an expression vector, most commonly a plasmid. A plasmid is a small, circular piece of DNA that replicates independently from the bacterium’s main chromosome. Scientists insert the gene of interest into the plasmid, creating what is known as a recombinant plasmid.

A promoter on the plasmid acts as an on/off switch for gene activity. Placed before the gene of interest, it controls when and how much protein is made. Inducible promoters are often used, which can be activated by a specific chemical. This control prevents protein production until the bacterial population has grown sufficiently.

The plasmid also contains a selectable marker, which is a gene providing resistance to a specific antibiotic. This feature ensures that only bacteria that have successfully taken up the plasmid can survive when exposed to that antibiotic. This allows for the isolation of successfully modified bacteria.

The Step-by-Step Expression Process

The first step is introducing the plasmid into E. coli cells, a procedure called transformation. A common method is heat shock, where bacteria are treated with calcium chloride to make their cell membranes permeable. The mixture of cells and plasmids is then briefly exposed to a high temperature, which encourages the plasmids to enter the cells.

After transformation, the bacteria are spread on an agar plate containing an antibiotic. As established, only bacteria that incorporated the plasmid with its resistance gene will grow and form colonies. This selection phase ensures the culture is composed purely of modified cells.

A successful colony is then grown in a large liquid culture. Protein production is initiated through induction, which activates the promoter controlling the gene. For example, a molecule like Isopropyl β-D-1-thiogalactopyranoside (IPTG) is added to the culture, acting as an inducer that turns on gene expression.

Once the promoter is activated, the cell’s machinery transcribes the gene’s DNA into messenger RNA (mRNA). The cell’s ribosomes then use the mRNA to synthesize the target protein. This process repeats, leading to the accumulation of the protein inside each cell until they are harvested.

Harvesting and Purifying the Protein

After production, the cells are broken open to release the protein in a process called cell lysis. The bacterial cell wall can be disrupted using physical methods, like sonication, or chemical methods involving detergents. This releases the cell’s contents, including the target protein.

The resulting mixture, or cell lysate, contains the target protein along with thousands of native bacterial proteins and other cellular debris. A purification process is required to isolate the target protein from this complex mixture based on its unique properties.

A common purification technique is affinity chromatography. This method involves engineering the protein with a small “affinity tag,” like a His-tag. The cell lysate is passed through a column containing beads that specifically bind to this tag. The tagged protein sticks to the beads while all other components wash away, isolating a pure protein sample.

Applications in Science and Medicine

This technology has significantly impacted medicine, especially in creating therapeutic proteins. A primary example is the production of human insulin to treat diabetes. Previously, insulin was extracted from animals, which was less effective and could cause allergic reactions. Bacterially-produced human insulin now provides a safe, reliable treatment, and this method is also used for vaccines and human growth hormone.

Bacterial protein expression is also used in industrial processes to generate enzymes. For instance, proteases and lipases are used in laundry detergents to break down protein and fat-based stains. In the food industry, the enzyme chymosin for cheese making is now produced in bacteria instead of being extracted from calf stomachs.

In scientific research, this technology provides the large quantities of pure protein needed to study structure and function. Obtaining these amounts from natural sources is often difficult or impossible. This capability allows researchers to investigate disease mechanisms and develop new drugs.